The aim of this project is to elucidate the factors that regulate lipid accumulation within mammalian cells, particularly the role of fatty acid saturation in this process. Emphasis will be placed on membrane function and intracellular cholesterol metabolism. We have recently succeeded in solubilizing acyl coenzyme A: cholesterol acyltransferase ACAT), a microsomal enzyme that catalyzes the synthesis of cholesteryl esters intracellularly. Attempts will be made to purify the solubilized ACAT activity. When a suitable degree of purification is achieved, the ACAT preparation will be combined with phospholipid vesicles. The aim of this work is to utilize the reconstituted system to gain further insight into the effects of phospholipid composition and phospholipid fatty acyl group composition on ACAT activity. This work will test the hypothesis that membrane lipid composition can exert a regulatory effect on ACAT activity. Additional work will be done in intact cells and microsomal preparations in order to determine whether all of the cholesterol in the microsomal membrane is accessible to ACAT. Preliminary work suggests that there is some compartmentalization of the membrane cholesterol with respect to availability as a substrate for ACAT, and this observation will be explored further. Membrane transport as a function of membrane lipid composition also will be examined. This will be done in intact Ehrlich ascites cells in which the fatty acid composition is modified by feeding different lipid-containing diets to the mice in which the cells are grown. In other experiments, vesicles will be prepared from Ehrlich cells and their properties in terms of transport investigated. Attempts will be made to modify the lipid composition of the vesicles by incubation with either liposomes or fatty acids. The transport properties of membrane vesicles having different lipid modifications will be compared.